Methods for improving organ viability

Abstract

This disclosure provides a method for the continuous administration of NO directly to one or more organs. x A method for improving the viability of one or more organs using a gas composition. A method for improving the viability of an organ intended for transplantation, the method comprising the continuous direct administration of a gas composition containing NO to the organ via an organ perfusion system or ventilation. x A composition of gases.

Description

[0001] This application is a divisional application of Chinese patent application No. 201880059363.9 (filed on August 24, 2018, invention title: Method for improving organ viability).

[0002] Cross-references to related applications

[0003] This application claims priority to U.S. Provisional Application No. 62 / 550,463, filed August 25, 2017, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0004] This disclosure provides a method for the continuous administration of NO directly to one or more organs. x A method of using gaseous compositions to enhance the viability of one or more organs. Background Technology

[0005] Cells, tissues, organs, and organisms that lose proper blood flow suffer ischemic injury due to oxidative stress and eventually die. Traditional methods for reducing ischemic injury involve perfusing the affected tissues with oxygen, but this can lead to significant tissue damage and may result in serious and / or permanent damage, such as brain injury during a stroke or cardiac arrest.

[0006] Attempts have been made to reduce ischemia and reperfusion injury by inducing tissues and organs into a reduced metabolic state. For example, in the case of preserving living tissue for transplantation, a common method to reduce tissue metabolic activity is to immerse the tissue or organ in a physiological fluid such as saline and place it in a cryogenic environment. However, such methods cannot be relied upon for extended periods, and the success of organ transplantation and limb reattachment remains inversely proportional to the time it takes for the organ or limb to be removed from the intact organism.

[0007] Therefore, there remains a need in the field for organs with improved survival prior to ischemia and / or reperfusion injury. Summary of the Invention

[0008] In one aspect, this disclosure includes a method for improving the viability of an organ intended for transplantation, the method comprising the continuous direct administration of NO-containing compounds to the organ via an organ perfusion system or ventilation. x A composition of gases.

[0009] In another aspect, this disclosure includes a method for improving the viability of organs damaged by ischemia-reperfusion, the method comprising the direct and continuous administration of NO containing no more than about 20 ppm directly to the organ via an organ perfusion system or ventilation. xA composition of gases. In various embodiments, the organ requiring treatment is an organ damaged due to traumatic injury, surgery, respiratory arrest, or cardiac arrest. In some embodiments, the organ requiring treatment is an organ intended for transplantation. In an exemplary embodiment, the organ requiring treatment is an organ removed from a donor for transplantation.

[0010] In another aspect, this disclosure includes a method for improving the viability of organs damaged by ischemia-reperfusion, the method comprising (a) administering to the organ NO containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), and then (b) continuously administered directly to the organ via an organ perfusion system or ventilation, containing no more than about 20 ppm of NO. x A composition of gases. In various embodiments, the organ requiring treatment is an organ damaged due to traumatic injury, surgery, respiratory arrest, or cardiac arrest. In some embodiments, the organ requiring treatment is an organ intended for transplantation. In an exemplary embodiment, the organ requiring treatment is an organ removed from a donor for transplantation.

[0011] In another aspect, this disclosure includes a method for improving the viability of organs intended for transplantation, the method comprising the direct and continuous administration of NO containing 20 ppm or less to the organ via an organ perfusion system or ventilation. x A composition of gases. In some embodiments, the organ is present in a brain-dead donor. In other embodiments, the gas is administered containing NO. x Prior to the composition of the gas, the organ has been removed from the donor. In an exemplary embodiment, the organ is a lung, kidney, or heart.

[0012] In another aspect, this disclosure includes a method for improving the viability of an organ intended for transplantation, the method comprising (a) administering to the organ a solution containing about 20 ppm to about 40 ppm of NO. x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), followed by (b) continuous direct administration of NO containing 20 ppm or less to the organ via an organ perfusion system or ventilation. x A composition of gases. In some embodiments, the organ is present in a brain-dead donor. In other embodiments, the gas is administered containing NO. x Prior to the composition of the gas, the organ has been removed from the donor. In an exemplary embodiment, the organ is a lung, kidney, or heart.

[0013] In another aspect, this disclosure includes a method for treating ischemia-reperfusion injury in organs requiring treatment, the method comprising the direct and continuous administration of NO containing 20 ppm or less to the organ via an organ perfusion system or ventilation. x A composition of gases. In various embodiments, the organ requiring treatment is an organ damaged due to traumatic injury, surgery, respiratory arrest, or cardiac arrest. In some embodiments, the organ requiring treatment is an organ intended for transplantation. In an exemplary embodiment, the organ requiring treatment is an organ removed from a donor for transplantation.

[0014] In another aspect, this disclosure includes a method for treating ischemia-reperfusion injury in an organ in need of treatment, the method comprising (a) administering to the organ NO containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), followed by (b) continuous direct administration of NO containing 20 ppm or less to the organ via an organ perfusion system or ventilation. x A composition of gases. In various embodiments, the organ requiring treatment is an organ damaged due to traumatic injury, surgery, respiratory arrest, or cardiac arrest. In some embodiments, the organ requiring treatment is an organ intended for transplantation. In an exemplary embodiment, the organ requiring treatment is an organ removed from a donor for transplantation.

[0015] In another aspect, this disclosure provides a method for transplantation, the method comprising (a) continuously administering directly to an organ intended for transplantation a substance containing 20 ppm or less NO. x The gas composition is administered for up to 12 hours, and (b) the organ is transplanted into the recipient. In some embodiments, the organ is present in a brain-dead donor. In other embodiments, the gas composition is administered with NO. x Prior to the composition of the gas, the organ has been removed from the donor. In an exemplary embodiment, the organ is a lung, kidney, or heart.

[0016] In another aspect, this disclosure provides a method for transplantation, the method comprising (a) administering to an organ NO containing about 20 ppm to about 40 ppm x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), followed by (b) continuous administration of NO containing 20 ppm or less directly to the organ to be transplanted. x The gaseous composition is used for up to 12 hours, and then (c) the organ is transplanted into the recipient. In some embodiments, the organ is present in a brain-dead donor. In other embodiments, the gaseous composition is administered with NO. xPrior to the composition of the gas, the organ has been removed from the donor. In an exemplary embodiment, the organ is a lung, kidney, or heart.

[0017] Other aspects and repetitions of this disclosure are described more fully below. Attached Figure Description

[0018] This application contains at least one color photograph. A copy of the published color photograph of this patent application will be provided by the office upon request and payment of the necessary fees.

[0019] Figure 1 This is a diagram illustrating one implementation of an EVLP circuit.

[0020] Figure 2 This is a diagram illustrating one implementation of an EVLP circuit. Detailed Implementation

[0021] This disclosure provides a method for the continuous administration of NO directly to one or more organs. x Methods for using gas compositions to enhance the viability of one or more organs. This disclosure includes methods for enhancing the viability of organs intended for transplantation and organs suffering from ischemia-reperfusion injury due to other causes. This disclosure also provides methods for improving the performance of transplanted organs and methods for transplantation. As used herein, the term "NO" refers to... x "Gas" refers to gaseous nitrogen oxides. In a preferred embodiment, NO... x The gas is gaseous nitric oxide (gNO). Non-limiting examples of other components of the composition may include an inert gas for dilution (e.g., helium, neon, etc.), human serum albumin, sodium caprylate, N-acetyl-DL-tryptophan, and oxygen (O2). Direct, continuous administration of NO to organs... x The composition of gases means that the organ will be continuously exposed to NO during administration. x Direct contact with the gas. This disclosure is not limited to organ type. Non-limiting examples of suitable organs include liver, kidney, pancreas, heart, lung, intestine, thymus, cornea, vascularized composite allogeneic grafts (e.g., face, hand, etc.), or any combination thereof. This is in contrast to continuous administration of NO through non-direct application to the organ. x Organs obtained by methods using gaseous compositions will have improved viability compared to those obtained by other methods. As used herein, the term "viability" refers to the suitability of an organ for its intended purpose. Measurements of organ viability can vary depending on organ type and are known in the art.

[0022] Several definitions applicable throughout this disclosure are presented below. As used herein, “about” means a numerical value, including integers, fractions, percentages, etc., whether or not explicitly indicated. The term “about” generally refers to a range of numerical values ​​that people would consider equivalent to the listed values ​​(e.g., having the same function or result), such as ±0.5-1%, ±1-5%, or ±5-10% of the listed values.

[0023] The term “comprising” means “including but not limited to”; specifically, it indicates open-ended inclusion or membership in said combination, group, series, etc. As used herein, the terms “comprising” and “including” are inclusive and / or open-ended and do not exclude additional, unlisted elements or methods. The term “substantially constitutes” is more restrictive than “comprising” but less binding than “consisting of.” Specifically, the term “substantially constitutes” limits membership to the specified materials or steps and those materials or steps that do not substantially affect the essential features of the claimed invention.

[0024] As used herein, the term “ischemia-reperfusion injury” refers to injury caused by ischemia, reperfusion, or both.

[0025] As used herein, the term "treatment" ("treat", "treating", or "treatment") refers to both therapeutic treatment and preventative or avoidant measures aimed at preventing or slowing (reducing) undesirable physiological changes or diseases / conditions. Beneficial or desired clinical outcomes include, but are not limited to, relief of symptoms, reduction of disease severity, stabilization of the disease state (i.e., no worsening), slowing or decelerating disease progression, improvement or mitigation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "Treatment" may also mean extending survival compared to expected survival without treatment. Individuals in need of treatment include those who already have a disease, symptom, or condition, those who are susceptible to a disease, symptom, or condition, or those who wish to prevent a disease, symptom, or condition.

[0026] (a) Contains NO x Gas composition

[0027] According to this disclosure, direct and continuous administration of NO to organs x Gas compositions. The compositions disclosed herein may be gases or liquids. When the compositions disclosed herein are liquids, NO... x The gas dissolves in the liquid. In other words, it "contains NO". x A gaseous composition, wherein the composition is a liquid, contains dissolved NO. x A liquid containing gas. Similarly, "containing 20 ppm NO" xThe gaseous composition, wherein the composition is a liquid, is a liquid containing 20 ppm nitric oxide, wherein the 20 ppm nitric oxide is NO dissolved in the liquid. x The amount of gas. Furthermore, in embodiments where the composition is a liquid, NO is applied directly to the organ. x The amount of gas is NO dissolved in the liquid. x The amount of gas. NO x The gas can be produced and supplied by any method known in the art.

[0028] In some embodiments, the composition is a gas. (Except for NO) x In addition to gases, the composition may also contain one or more additional components, including but not limited to inert gases for dilution (e.g., helium, neon, etc.), nitrogen, oxygen, and water. In one exemplary embodiment, NO x The gas is gNO. When the composition is a gas, it can be administered directly to organs via a ventilator or any other method known in the art.

[0029] In some embodiments, the composition is an perfusion solution. The term "perfusion solution" refers to any fluid used for the preservation, perfusion, or reperfusion of tissues or organs. Perfusion solutions are typically sterile and isotonic. Except for dissolved NO... x In addition to the gas, the perfusion fluid may also contain one or more additional components, including but not limited to sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. The composition of the perfusion fluid may also vary depending on the organ. In a preferred embodiment, the perfusion fluid is a cell-free perfusion fluid. Such solutions may include, but are not limited to, Celsior solution, Krebs-Henseleit solution, physiological saline, University of Wisconsin solution, St. Thomas II solution, Collins solution, Stanford solution, Perfidex®, Steen Solution™, or combinations thereof. In an exemplary embodiment, the composition is a cell-free perfusion fluid, and NO x The gas is gNO. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. Suitable methods for the direct administration of perfusion solutions to organs are known in the art, including but not limited to organ perfusion systems. This disclosure is not limited to any particular organ perfusion system. Generally, organ perfusion systems may include pumps for perfusion motion and control, devices for controlling system temperature, cannulas, and devices for measuring physiological parameters. Non-limiting examples of organ perfusion systems are disclosed in U.S. Patent No. 9,629,358, which is incorporated herein by reference. Figure 1 and Figure 2The paper also discloses non-limiting examples of organ perfusion systems.

[0030] To improve organ viability, a therapeutically effective dose of NO is directly administered to the organ. x Gas. Depending on the type of composition applied, NO is administered directly to the organ. x The gas can be delivered via an organ perfusion system, a ventilator, or any combination thereof. In embodiments that combine an organ perfusion system and a ventilator, the organ perfusion system and the ventilator can be used simultaneously to directly administer NO to the organ. x A gaseous composition. Alternatively or in addition, an organ perfusion system and a ventilator may be used sequentially to administer NO-containing compounds directly to organs. x The composition of gases includes different amounts of overlap between two administration methods (e.g., no overlap or overlap of seconds, minutes, or hours). For example, administration may be performed first with a ventilator and then with an infusion system, or vice versa.

[0031] NO x "Therapeutic effective dose of gas" refers to the amount of NO that, when administered directly to an organ, is sufficient to improve the viability of the organ as defined in this article. x The amount of gas. NO that constitutes the "therapeutic effective dose". x The amount will vary depending on various factors, but can be determined by those skilled in the art. As further detailed below, NO is used to treat organs with ischemia-reperfusion injury. x The therapeutically effective dose of the gas is 20 ppm or less. This therapeutically effective dose of NO... x The gas can be used alone or in the application of a loading dose of NO. x Use after gas administration. Loading dose of NO x The gas can be used to increase vasodilation in ischemic organs and is particularly suitable when the compositions of this disclosure first come into contact with the organ after removal from the donor. However, the NO provided in the loading dose... x The amount of gas is typically greater than that used for the safe treatment of ischemia-reperfusion injury over a prolonged period (e.g., a period exceeding one hour). x The amount of gas.

[0032] In one or more implementations, NO xThe gas is applied at an initial concentration and optionally increased as needed to achieve the desired effect (e.g., increased organ viability). For example, the initial nitric oxide concentration may be from about 0.05 ppm to about 50 ppm or from about 1 ppm to about 50 ppm, and optionally increased until the desired effect is achieved or the nitric oxide threshold is reached. An exemplary nitric oxide application may begin with an initial concentration of about 1 ppm, and then increase in increments of about 0.1 ppm to about 5 ppm until the desired NO is achieved. x Effect, but ensure NO x The concentration should not exceed 50 ppm and / or the methemoglobin level should not reach or exceed 5%. Alternatively, exemplary nitric oxide application may begin with an initial concentration of 5 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 50 ppm and / or the methemoglobin level does not reach or exceed 5%. In another exemplary embodiment, nitric oxide application may begin with an initial concentration of 10 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 50 ppm and / or the methemoglobin level does not reach or exceed 5%. In yet another exemplary embodiment, nitric oxide application may begin with an initial concentration of 15 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 50 ppm and / or the methemoglobin level does not reach or exceed 5%. In yet another exemplary embodiment, nitric oxide application may begin with an initial concentration of 20 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration shall not exceed 50 ppm and / or the methemoglobin level shall not reach or exceed 5%. In each of the above embodiments, the application may be sustained for 5, 10, 15, 30 or 60 minutes. Alternatively, the application may be sustained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0033] In one or more implementations, NO xThe gas is applied at an initial concentration and optionally increased as needed to achieve the desired effect (e.g., increased organ viability). For example, the initial nitric oxide concentration may be from about 0.05 ppm to about 35 ppm or from about 1 ppm to about 35 ppm, and optionally increased until the desired effect is achieved or the nitric oxide threshold is reached. An exemplary nitric oxide application may begin with an initial concentration of about 1 ppm, and then increase in increments of about 0.1 ppm to about 5 ppm until the desired NO is achieved. x Effect, but ensure NO x The concentration should not exceed 35 ppm and / or the methemoglobin level should not reach or exceed 5%. Alternatively, exemplary nitric oxide application may begin with an initial concentration of 5 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 35 ppm and / or the methemoglobin level does not reach or exceed 5%. In another exemplary embodiment, nitric oxide application may begin with an initial concentration of 10 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 35 ppm and / or the methemoglobin level does not reach or exceed 5%. In yet another exemplary embodiment, nitric oxide application may begin with an initial concentration of 15 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 35 ppm and / or the methemoglobin level does not reach or exceed 5%. In yet another exemplary embodiment, nitric oxide application may begin with an initial concentration of 20 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration shall not exceed 35 ppm and / or the methemoglobin level shall not reach or exceed 5%. In each of the above embodiments, the application may be sustained for 5, 10, 15, 30 or 60 minutes. Alternatively, the application may be sustained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0034] In one or more implementations, NO xThe gas is applied at an initial concentration and optionally increased as needed to achieve the desired effect (e.g., increased organ viability). For example, the initial nitric oxide concentration may be from about 0.05 ppm to about 20 ppm or from about 1 ppm to about 20 ppm, and optionally increased until the desired effect is achieved or the nitric oxide threshold is reached. An exemplary nitric oxide application may begin with an initial concentration of about 1 ppm, and then increase in increments of about 0.1 ppm to about 5 ppm until the desired NO is achieved. x Effect, but ensure NO x The concentration should not exceed 20 ppm and / or the methemoglobin level should not reach or exceed 5%. Alternatively, exemplary nitric oxide application may begin with an initial concentration of 5 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 20 ppm and / or the methemoglobin level does not reach or exceed 5%. In another exemplary embodiment, nitric oxide application may begin with an initial concentration of 10 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 35 ppm and / or the methemoglobin level does not reach or exceed 5%. In yet another exemplary embodiment, nitric oxide application may begin with an initial concentration of 15 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration does not exceed 20 ppm and / or the methemoglobin level does not reach or exceed 5%. In yet another exemplary embodiment, nitric oxide application may begin with an initial concentration of 20 ppm, then increase in increments of 0.1 ppm to 5 ppm until the desired NO is obtained. x Effect, but ensure NO x The concentration shall not exceed 20 ppm and / or the methemoglobin level shall not reach or exceed 5%. In each of the above embodiments, the application may be sustained for 5, 10, 15, 30 or 60 minutes. Alternatively, the application may be sustained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 hours.

[0035] In one or more implementations, NO x The gas is applied at an initial concentration and for an initial time, followed by a second application at a lower concentration and for a second time to achieve the desired effect (e.g., increased organ viability). For example, NO... xThe gas can be administered at an initial concentration of about 20 ppm to about 40 ppm for up to about 1 hour, and then reduced to 20 ppm or lower to improve organ viability. Exemplary nitric oxide administration can begin with an initial concentration of about 20 ppm to about 40 ppm, and then decrease over an initial time period until the nitric oxide concentration is 20 ppm or lower. The rate of reduction may be constant or not. The nitric oxide concentration may also be adjusted as needed (e.g., based on monitoring of nitric oxide markers). Alternatively, exemplary nitric oxide administration can begin with an initial concentration of about 20 ppm to about 40 ppm, remain constant over an initial time period, and then decrease to a nitric oxide concentration of 20 ppm or lower. The nitric oxide concentration may also be adjusted as needed (e.g., based on monitoring of nitric oxide markers). In each of the above embodiments, the total time of nitric oxide administration (time for initial concentration plus time for concentration reduction) may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours.

[0036] In each of the above embodiments, the initial nitric oxide concentration, the increment of nitric oxide increase or decrease, the maximum nitric oxide concentration, and / or the threshold for nitric oxide or nitric oxide markers may vary depending on the application and / or based on the specific organ being treated. The increment may vary throughout the adjustment of nitric oxide delivery. Nitric oxide may also be decremented if monitoring indicates that nitric oxide or nitric oxide markers have reached or exceeded the nitric oxide threshold.

[0037] In each of the above implementation schemes, NO x Concentration can also be calculated relative to the final NO x The concentration is adjusted in increments of a certain percentage. These percentage increments can include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, and 200% of NO. x Concentration changes.

[0038] Instead of adjusting NO x Concentration or adjustment of NO x In addition to concentration, NO can be adjusted by regulating its direct delivery to organs. x Any method of adjusting the amount of gas (such as by adjusting NO) x Adjusting the NO flow rate x Gas application.

[0039] In other embodiments, nitric oxide administration is adjusted based on monitoring of nitric oxide or nitric oxide markers. As used herein, "nitric oxide marker" refers to a direct or indirect indicator of nitric oxide concentration in a liquid. Examples of nitric oxide markers include methemoglobin and NO. x (i.e., NO, nitrite ions (NO2) − ), nitrate ions (NO3) − (etc.) and others. This adjustment can be manual or by NO. x The delivery system is automated. The NO delivery system can also provide alerts based on monitoring. If the monitoring device is a separate component from the NO delivery system, it can transmit monitoring information to the NO delivery system via any suitable wired or wireless connection. For example, if the level of nitric oxide or nitric oxide markers in the liquid falls below a certain threshold, NO delivery can be increased until the level reaches the threshold. Similarly, if the level of nitric oxide or nitric oxide markers in the liquid exceeds a certain threshold, the amount of NO applied can be reduced. Continuous or intermittent monitoring of nitric oxide or nitric oxide markers can be performed, such as periodic monitoring.

[0040] In one or more embodiments, such monitoring may include monitoring methemoglobin and / or NO. These nitric oxide markers can be measured directly by techniques such as pulse oximetry or optical measurements, or any other means for directly or indirectly measuring or correlating NO or NO markers. For example, another measurement technique involves placing a probe in the perfusion fluid to measure liquid NO. x It provides a level and can offer real-time analysis of the perfusion fluid.

[0041] In one or more embodiments, nitric oxide or nitric oxide markers are monitored by comparing a measured value of nitric oxide or a nitric oxide marker to a nitric oxide threshold. The nitric oxide threshold can be a safety limit to ensure that methemoglobinemia does not develop. For example, the nitric oxide threshold can be a methemoglobin level, such as the percentage of methemoglobin relative to red blood cells. In exemplary embodiments, the nitric oxide threshold is in the range of about 1% to about 15% methemoglobin or about 3% to about 10% methemoglobin. Therefore, if the methemoglobin level reaches or exceeds an acceptable range, such as ≤3%, ≤4%, ≤5%, ≤6%, ≤7%, ≤8%, ≤9%, ≤10%, ≤11%, or ≤12%, nitric oxide administration can be adjusted.

[0042] NO2 levels can also be monitored in the infusion solution. Due to liquid recirculation, NO2 can accumulate in the liquid. If the NO2 concentration rises above a certain threshold, the NO delivery device can adjust NO application and / or provide an alarm. NO2 can also be removed by using reducing agents, scrubbers, alkalis, or other suitable methods.

[0043] (b) Ischemia-reperfusion injury

[0044] The term "ischemia-reperfusion injury" refers to injury resulting from ischemia, reperfusion, or both. Ischemia refers to insufficient blood supply to an organ, and ischemic injury occurs when the blood supply to a region of tissue or organ is interrupted. The act of restoring blood flow to an organ or tissue is called reperfusion, and reperfusion injury occurs due to the restoration of blood flow to a tissue or organ after ischemia. Ischemia can be the result of injury or disease to an organism. Examples of specific diseases that can cause ischemia or hypoxia include, but are not limited to, traumatic injuries or surgery, respiratory or cardiac arrest, tumors, heart disease, and neurological disorders. Examples of specific injuries that can lead to ischemic or hypoxic conditions include, but are not limited to, external injuries such as burns, lacerations, amputations, gunshot wounds, or surgical trauma. In addition, injury can also include internal injuries such as stroke or heart attack, which result in a sharp reduction in blood circulation. Other injuries include reduced blood circulation due to non-invasive stress (such as exposure to cold or radiation) or planned reduction in blood circulation (e.g., during cardiac surgery or in treatments performed on an organ donor before removal for transport and transplantation to a recipient).

[0045] One aspect of this disclosure includes a method for treating ischemia-reperfusion injury in organs requiring treatment. The method involves the direct and continuous administration of NO containing 20 ppm or less directly to the organ. x A gaseous composition. In some embodiments, the composition contains 1 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 ppm to about 10 ppm of nitric oxide, about 5 ppm to about 15 ppm of nitric oxide, or about 10 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm of nitric oxide. Contains NO. xSuitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, and even more preferably a cell-free perfusion solution. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. In various embodiments, the organ requiring treatment is an organ damaged due to traumatic injury, surgery, respiratory arrest, or cardiac arrest. In some embodiments, the organ requiring treatment is an organ intended for transplantation. In an exemplary embodiment, the organ requiring treatment is an organ removed from a donor for transplantation.

[0046] Application can last for 5, 10, 15, 30, or 60 minutes. Alternatively, application can last for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours. In implementation schemes where the organ is intended for transplantation, NO x Gas administration preferably does not exceed a total of 12 hours. In some embodiments, administration begins simultaneously with ischemia. In other embodiments, administration begins sometime after the onset of ischemia, but preferably as close as possible to the onset of ischemia. For example, administration may begin approximately 5, 10, 15, 20, 25, or 30 minutes after the onset of ischemia. Administration may also begin during reperfusion, or alternatively, continue after the onset of reperfusion. In some cases, administration may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 hours after the onset of reperfusion.

[0047] Effective treatment of ischemia-reperfusion injury can be assessed by any method known in the art, including but not limited to measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological assessment, protein oxidation, morphological changes, etc.), measuring inflammation, and / or measuring organ function.

[0048] In other embodiments, a method for treating ischemia-reperfusion injury in an organ requiring treatment may include an additional step of administering NO containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), immediately followed by the administration of NO containing 20 ppm or less. xA composition of gases. For example, a loading dose may be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 10 minutes to about 30 minutes. In another example, a loading dose may be administered for about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or 30 minutes to 60 minutes. The two compositions may be the same except for the nitric oxide concentration. Alternatively, the two compositions may be different. The concentration of nitric oxide in the loading dose may be decreased during the loading period until the nitric oxide concentration is 20 ppm or lower. The rate of decrease may be constant or not constant. Alternatively, the concentration of nitric oxide in the loading dose may be kept constant during the loading period and then decreased to a nitric oxide concentration of 20 ppm or lower. In a preferred embodiment, the two compositions are the same, and the composition is a perfusion fluid, preferably a cell-free perfusion fluid. In an exemplary embodiment, the organ is a heart, lung, or kidney, and the organ is intended for transplantation.

[0049] Another aspect of this disclosure includes methods for improving the survival of organs damaged by ischemia-reperfusion injury. The methods involve the direct and continuous administration of NO containing 20 ppm or less to the organ. x A gaseous composition. In some embodiments, the composition contains 1 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 ppm to about 10 ppm of nitric oxide, about 5 ppm to about 15 ppm of nitric oxide, or about 10 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm of nitric oxide. Contains NO. x Suitable compositions of gases are described in section (a). In a preferred embodiment, the composition is a perfusion fluid, and even more preferably a cell-free perfusion fluid. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. Improving the viability of organs damaged by ischemia and / or reperfusion may include preserving mitochondrial function or reducing oxidative damage. Other methods known in the art for assessing organ viability may also be used, including but not limited to measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological assessment, morphological changes, etc.), measuring inflammation, and / or measuring organ function.

[0050] In one embodiment, this disclosure includes a method for preserving mitochondrial function in an organ with ischemia-reperfusion injury. As used herein, mitochondrial function can be measured by respiratory control rate (RCR), an indicator of mitochondrial coupling status. Generally, RCR represents the ratio of the oxidation rate in the presence of excess substrate and adenosine diphosphate (ADP) (state 3) to the oxidation rate after ADP phosphorylation to a steady-state concentration (state 4). In some embodiments, this is achieved by directly administering NO containing 20 ppm or less to the organ. x A gaseous composition significantly preserves mitochondrial function in organs with ischemia-reperfusion injury. As used herein, "significantly preserves" means, as described herein, mitochondrial function preserved by NO. x The difference in mitochondrial function between organs treated with gas and control organs that did not experience ischemia-reperfusion was less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or less than 95%. In other words, significant preservation could be defined as the difference between organs treated with gas and those that experienced similar ischemia-reperfusion injury but were not directly and continuously treated with NO. x Compared to similar organs containing gas, mitochondrial function is improved. (Contains NO) x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0051] In other embodiments, this disclosure includes methods for reducing oxidative damage to organs with ischemia-reperfusion injury. Generally, the method involves the direct, continuous administration of NO containing 20 ppm or less to the organ. x A composition of gases. As used herein, “reduced oxidative damage” or “reduced oxidative damage” can be associated with treatment under similar conditions, but without direct and continuous administration of NO. x The measurement is made by comparing the organs to which the gas is being administered. For example, compared to those treated under similar conditions but not directly and continuously given NO. x Compared to organs treated with NO, oxidative damage was reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In a specific implementation, compared to an untreated control, mitochondrial reactive oxygen species (mtROS) were reduced after direct and continuous NO administration. x Reduced levels in organs with ischemia-reperfusion injury. Contains NO. xSuitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0052] In some embodiments, this disclosure includes a method for increasing superoxide dismutase 2 (SOD2 or manganese-dependent superoxide dismutase (MnSOD)) activity in organs with ischemia-reperfusion injury. The method includes the direct, continuous administration of a solution containing 20 ppm or less of NO directly to the organ. x A gaseous composition wherein MnSOD activity is increased in said organ compared to a control organ not exposed to the compositions of this disclosure. For example, compared to treatment under similar conditions but without direct and continuous administration of NO. x Compared to organs containing gases, MnSOD activity can be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Methods for measuring MnSOD activity are known in the art. (Contains NO) x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0053] In other embodiments, this disclosure includes a method for inhibiting the formation of nitrotyrosine in organs with ischemia-reperfusion injury. The method includes continuous direct administration of NO containing 20 ppm or less to the organ. x A gaseous composition in which the formation of nitrotyrosine adducts is inhibited in a control organ, compared to an organ not exposed to the composition of the present invention. For example, compared to treatment under similar conditions, but without direct and continuous administration of NO. x Compared to gaseous organs, nitrotyrosine formation can be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Methods for measuring the formation of nitrotyrosine adducts are known in the art. (Contains NO) xSuitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0054] In some embodiments, this disclosure includes a method for preventing the inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or a combination thereof in an organ with ischemia-reperfusion injury. The method includes the direct, continuous administration of NO containing 20 ppm or less to the organ. x A gaseous composition wherein 20 ppm or less of NO was administered compared to a control organ. x The gas prevents the inactivation of mitochondrial complexes I, II, III, IV, or combinations thereof. In one embodiment, a continuous direct administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex I. In another embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex II activity. In yet another embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex III activity. In another embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex IV activity. In a preferred embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex I and mitochondrial complex II. In another preferred embodiment, direct and continuous administration of NO containing 20 ppm or less is performed into the organ. x The gaseous composition prevents the inactivation of mitochondrial complex II and mitochondrial complex III. For example, in treatment under similar conditions, but without direct and continuous administration of NO... x Compared to the gaseous organs, the inactivation of activity in each of the above embodiments can be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Methods for measuring the activity of mitochondrial complex I, complex II, complex III, or complex IV are known in the art. Containing NO xSuitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0055] In each of the above embodiments, NO is contained in 20 ppm or less. x The gaseous composition can be administered for a duration necessary to enhance organ viability. For example, in some embodiments, administration can be sustained for 5, 10, 15, 30, or 60 minutes. In other embodiments, administration can be sustained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours. In embodiments where the organ is intended for transplantation, NO x Gas administration preferably does not exceed a total of 12 hours. In some embodiments, administration begins simultaneously with ischemia. In other embodiments, administration begins at some point after the onset of ischemia, but preferably as close as possible to the onset of ischemia. In some embodiments, administration begins approximately 5, 10, 15, 20, 25, or 30 minutes after the onset of ischemia. Administration may also begin during reperfusion, or alternatively, continue after the onset of reperfusion. In some cases, administration may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more than 12 hours after the onset of reperfusion.

[0056] In other embodiments, the above method may include an additional step of applying NO containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour, immediately followed by the administration of NO containing 20 ppm or less. xA gaseous composition. For example, a loading dose may be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 10 minutes to about 30 minutes. In another example, a loading dose may be administered for about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or 30 minutes to 60 minutes. The two compositions may be the same except for the nitric oxide concentration. Alternatively, the two compositions may be different. The concentration of nitric oxide in the loading dose may decrease over a period of up to about one hour until the nitric oxide concentration is 20 ppm or lower. The rate of decrease may be constant or not. Alternatively, the concentration of nitric oxide in the loading dose may remain constant over a period of up to about one hour and then decrease to a nitric oxide concentration of 20 ppm or lower. In a preferred embodiment, the two compositions are the same, and the composition is an infusion solution, preferably a cell-free infusion solution. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. In an exemplary embodiment, the organ is the heart, lung, or kidney.

[0057] (c) Methods to improve the viability of organs used for transplantation

[0058] Organs with ischemia-reperfusion injury include organs intended for transplantation. Therefore, this disclosure includes methods for improving the viability of organs intended for transplantation. Such methods include the direct and continuous administration of NO-containing compounds to the organ via an organ perfusion system or ventilation. x The gaseous composition lasts for a maximum of 12 hours. In other words, from the time the organ is harvested until it is transplanted in the recipient, the organ will be continuously exposed to NO. x Direct contact with the gas. As used herein, acquisition refers to the identification of an organ donor and the removal of the organ, and may be used interchangeably with any of the terms. In some embodiments, the composition is administered after the organ has been obtained from the donor. In other embodiments, the composition is administered while the organ is still in the donor's body. In these embodiments, the donor may be a brain-dead donor or a donor without a heartbeat. In some instances, administration may last for 5, 10, 15, 30, or 60 minutes. In other instances, administration may last for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. Contains NO. xSuitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, and even more preferably a cell-free perfusion solution. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. Without wishing to be bound by theory, it is believed that the methods of this disclosure can increase the number of organs available for transplantation by improving the viability of organs that previously did not meet transplantation criteria, thus enabling the use of more organs from more donors (e.g., marginal brain-dead donors, heartless donors, etc.). Improving the viability of organs used for transplantation may, in part, involve preserving mitochondrial function or reducing oxidative damage in the organ. Other methods known in the art for assessing organ viability may also be used, including but not limited to measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological assessment, morphological changes, etc.), measuring inflammation, and / or measuring organ function. In an exemplary embodiment, the organ is a heart, lung, or kidney.

[0059] In one embodiment, this disclosure includes a method for preserving mitochondrial function in an organ intended for transplantation. As used herein, mitochondrial function can be measured by respiratory control rate (RCR), an indicator of mitochondrial coupling status. Generally, RCR represents the ratio of the oxidation rate in the presence of excess substrate and adenosine diphosphate (ADP) (state 3) to the oxidation rate after ADP phosphorylation to a steady-state concentration (state 4). In some embodiments, this is achieved by directly administering NO containing 20 ppm or less to the organ. x A gaseous composition significantly preserves mitochondrial function in organs intended for transplantation. As used herein, "significantly preserves" means, as described herein, the gaseous composition of NO... x Organs treated with gas and those not treated with NO x The differences in mitochondrial function among control organs treated with gas were less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or less than 95%. In other words, significant preservation could refer to patients who had experienced similar ischemia-reperfusion injury but were not directly and continuously treated with NO. x Compared to similar organs used for transplantation, the gaseous form showed improved mitochondrial function. (Contains NO) x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is SteenSolution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0060] In certain embodiments, this disclosure includes a method for reducing oxidative damage to organs intended for transplantation. For example, mitochondrial reactive oxygen species (mtROS) can be reduced in organs intended for transplantation. This method involves the direct, continuous administration of NO containing 20 ppm or less to the organ. x A gaseous composition. Direct, continuous application can occur before organ removal from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ will be continuously exposed to NO from the time of removal until transplantation in the recipient. x Direct contact with the gas. As used herein, "reduced oxidative damage" or "decreased oxidative damage" can be synonymous with treatment under similar conditions, but without direct and continuous administration of NO. x The measurement is made by comparing the organs to which the gas is being administered. For example, compared to those treated under similar conditions but not directly and continuously given NO. x Compared to organs treated with NOx, oxidative damage was reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In a specific implementation, mitochondrial reactive oxygen species (mtROS) were reduced in organs with ischemia-reperfusion injury after direct, continuous NOx administration, compared to untreated controls. (NO-containing...) x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0061] In some embodiments, this disclosure includes a method for increasing superoxide dismutase 2 (SOD2 or manganese-dependent superoxide dismutase (MnSOD)) activity in an organ intended for transplantation. The method includes the direct, continuous administration of a solution containing 20 ppm or less of NO directly to the organ. x A gaseous composition wherein MnSOD activity is increased in said organ compared to a control organ not exposed to the compositions of this disclosure. For example, compared to treatment under similar conditions but without direct and continuous administration of NO. xCompared to organs treated with gaseous NO, MnSOD activity can be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Direct, continuous administration can occur before organ removal from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ will be continuously exposed to NO from the time of removal until transplantation in the recipient. x Direct contact with the gas. Methods for measuring MnSOD activity are known in the art. Contains NO. x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0062] In other embodiments, this disclosure includes a method for inhibiting the formation of nitrotyrosine in an organ intended for transplantation. The method includes the direct and continuous administration of NO containing 20 ppm or less to the organ. x A gaseous composition wherein the formation of nitrotyrosine adducts is inhibited in said organ compared to a control organ not exposed to the compositions of this disclosure. For example, compared to treatment under similar conditions, but without direct and continuous administration of NO. x Compared to gaseous organs, the formation of nitrotyrosine can be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Direct, continuous administration can occur before organ removal from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ will be continuously exposed to NO from the time of removal until transplantation in the recipient. x Direct contact with the gas. Methods for measuring the formation of nitrotyrosine adducts are known in the art. Contains NO. x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0063] In some embodiments, this disclosure includes a method for preventing the inactivation of mitochondrial complex I activity, complex II activity, complex III activity, complex IV activity, or a combination thereof in an organ intended for transplantation. The method includes the direct, continuous administration of NO containing 20 ppm or less to the organ. x A gaseous composition wherein 20 ppm or less of NO was administered compared to a control organ. x The gas prevents the inactivation of mitochondrial complexes I, II, III, IV, or combinations thereof. In one embodiment, a continuous direct administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex I. In another embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex II activity. In yet another embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex III activity. In another embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex IV activity. In a preferred embodiment, direct, continuous administration of NO containing 20 ppm or less is performed onto the organ. x The gaseous composition prevents the inactivation of mitochondrial complex I and mitochondrial complex II. In another preferred embodiment, direct and continuous administration of NO containing 20 ppm or less is performed into the organ. x The gaseous composition prevents the inactivation of mitochondrial complex II and mitochondrial complex III. For example, in treatment under similar conditions, but without direct and continuous administration of NO... x Compared to the gaseous organ, the inactivation of the active component in each of the above embodiments can be inhibited by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Direct, continuous application can occur before organ removal from the donor, during transport / storage, during transplantation to the recipient, after transplantation to the recipient, or any combination thereof. In a preferred embodiment, the organ will be continuously exposed to NO from the time of removal until transplantation in the recipient. x Direct gas contact. Methods for measuring the activity of mitochondrial complex I, complex II, complex III, or complex IV are known in the art. Contains NO. xSuitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0064] In each of the above methods, NO is directly administered to the organ via an organ perfusion system. x The gas can be generated via an organ perfusion system, a ventilator, or any combination thereof, for a duration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In embodiments using a combination of an organ perfusion system and a ventilator, the organ perfusion system and the ventilator can be used simultaneously to directly administer NO to the organ. x A gaseous composition. Alternatively or in addition, an organ perfusion system and a ventilator may be used sequentially to administer NO-containing compounds directly to organs. x The composition of gases includes different amounts of overlap between two administration methods (e.g., no overlap or overlap of seconds, minutes, or hours). For example, administration may be performed first with a ventilator and then with an infusion system, or vice versa.

[0065] In other embodiments, the above method may include an additional step of applying NO containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour, immediately followed by the administration of NO containing 20 ppm or less. x A gaseous composition in which NO x The gas should not be administered for a total of more than 12 hours. For example, a loading dose can be administered over approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, or approximately 10 minutes to approximately 30 minutes, followed by administration of NO containing 20 ppm or less. x The gaseous composition lasts for approximately 11.8 hours or less. In another example, a loading dose may be administered for approximately 30 minutes, approximately 35 minutes, approximately 40 minutes, approximately 40 minutes, approximately 45 minutes, approximately 50 minutes, approximately 55 minutes, approximately 60 minutes, or 30 to 60 minutes, followed by administration of NO containing 20 ppm or less. xThe gaseous composition is used for 11.5 hours or less. The two compositions may be identical except for the nitric oxide concentration. Alternatively, the two compositions may be different. The concentration of nitric oxide in the loading dose may be decreased over a period of up to about one hour until the nitric oxide concentration is 20 ppm or lower. The rate of decrease may be constant or not. Alternatively, the concentration of nitric oxide in the loading dose may be kept constant over a period of up to about one hour and then decreased to a nitric oxide concentration of 20 ppm or lower. In a preferred embodiment, the two compositions are identical, and the composition is a perfusion solution, preferably a cell-free perfusion solution. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. In an exemplary embodiment, the organ is the heart, lung, or kidney.

[0066] (d) Methods to improve the post-transplant performance of organs used for transplantation

[0067] Another aspect of this disclosure includes methods for improving the post-transplant performance of organs used for transplantation. The methods involve the continuous direct administration of NO containing 20 ppm or less to the organ via an organ perfusion system. x The gaseous composition lasts for a maximum of 12 hours. In some instances, application can be sustained for 5, 10, 15, 30, or 60 minutes. In other instances, application can be sustained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. Contains NO. x Suitable compositions of gases are described in section (a). In a preferred embodiment, the composition is a perfusion fluid, and even more preferably a cell-free perfusion fluid. Improving the post-transplant performance of the organ to be transplanted may include half an inch of mitochondrial function or reducing oxidative damage. Other methods known in the art for assessing organ viability may also be used, including but not limited to measuring cellular function (e.g., metabolic capacity, ATP content, etc.), measuring cellular damage (e.g., histological assessment, morphological changes, etc.), measuring inflammation, and / or measuring organ function. In an exemplary embodiment, the organ is a heart, lung, or kidney.

[0068] In one embodiment, this disclosure includes a method for preserving mitochondrial function in a post-transplant organ. As used herein, mitochondrial function can be measured by respiratory control rate (RCR), an indicator of mitochondrial coupling status. Generally, RCR represents the ratio of the oxidation rate in the presence of excess substrate and adenosine diphosphate (ADP) (state 3) to the oxidation rate after ADP phosphorylation to a steady-state concentration (state 4). In some embodiments, this is achieved by directly administering NO containing 20 ppm or less to the organ. x The gaseous composition significantly preserves mitochondrial function in transplanted organs. As used herein, "significantly preserves" means, as described herein, the process involving NO... xOrgans treated with gas and those not treated with NO x The differences in mitochondrial function among control organs treated with direct continuous gas therapy were less than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or less than 95%. In other words, significant preservation could refer to patients who had experienced similar ischemia-reperfusion injury but were not directly and continuously treated with NO. x Compared to similar organs containing gas, mitochondrial function is improved. (Contains NO) x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0069] In other embodiments, this disclosure includes methods for reducing oxidative damage in post-transplant organs. Generally, the method involves the direct and continuous administration of NO containing 20 ppm or less to the organ. x A composition of gases. As used herein, “reduced oxidative damage” or “reduced oxidative damage” can be associated with treatment under similar conditions, but without direct and continuous administration of NO. x The measurement is made by comparing the organs to which the gas is being administered. For example, compared to those treated under similar conditions but not directly and continuously given NO. x Compared to organs treated with NOx, oxidative damage was reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In a specific implementation, mitochondrial reactive oxygen species (mtROS) were reduced in transplanted organs subjected to direct, continuous NOx administration compared to untreated controls. (NO-containing...) x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, even more preferably a cell-free perfusion solution, and the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0070] In other embodiments, the above method may include an additional step of applying NO containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour, immediately followed by the administration of NO containing 20 ppm or less. x A gaseous composition in which NOx The gas should not be administered for a total of more than 12 hours. For example, a loading dose can be administered over approximately 10 minutes, 15 minutes, 20 minutes, 30 minutes, or approximately 10 minutes to approximately 30 minutes, followed by administration of NO containing 20 ppm or less. x The gaseous composition lasts for approximately 11.8 hours or less. In another example, a loading dose may be administered for approximately 30 minutes, approximately 35 minutes, approximately 40 minutes, approximately 40 minutes, approximately 45 minutes, approximately 50 minutes, approximately 55 minutes, approximately 60 minutes, or 30 to 60 minutes, followed by administration of NO containing 20 ppm or less. x The gaseous composition is used for 11.5 hours or less. The two compositions may be identical except for the nitric oxide concentration. Alternatively, the two compositions may be different. The concentration of nitric oxide in the loading dose may be decreased over a period of up to about one hour until the nitric oxide concentration is 20 ppm or lower. The rate of decrease may be constant or not. Alternatively, the concentration of nitric oxide in the loading dose may be kept constant over a period of up to about one hour and then decreased to a nitric oxide concentration of 20 ppm or lower. In a preferred embodiment, the two compositions are identical, and the composition is a perfusion solution, preferably a cell-free perfusion solution. In an exemplary embodiment, the organ is the heart, lung, or kidney. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

[0071] (e) Methods used for transplantation

[0072] In another aspect, this disclosure provides a method for transplantation. The method includes (a) continuously administering a solution containing 20 ppm or less of NO directly to the organ to be transplanted. x The gaseous composition is administered for up to 12 hours, and (b) the organ is transplanted into the recipient. Administration can be sustained for 5, 10, 15, 30, or 60 minutes. Alternatively, administration can be sustained for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 hours. In some embodiments, the composition contains 1 ppm to 20 ppm of nitric oxide. In other embodiments, the composition contains about 1 ppm to about 10 ppm of nitric oxide, about 5 ppm to about 15 ppm of nitric oxide, or about 10 ppm to 20 ppm of nitric oxide. In other embodiments, the composition comprises about 1 ppm, about 2 ppm, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7 ppm, about 8 ppm, about 9 ppm, about 10 ppm, about 11 ppm, about 12 ppm, about 13 ppm, about 14 ppm, about 15 ppm, about 16 ppm, about 17 ppm, about 18 ppm, about 19 ppm, or about 20 ppm of nitric oxide. Contains NO.x Suitable compositions of the gas are described in section (a). In a preferred embodiment, the composition is a perfusion solution, and even more preferably a cell-free perfusion solution. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. In an exemplary embodiment, the organ is the heart, lung, or kidney.

[0073] In some embodiments, the organ to be transplanted has been removed from the donor prior to step (a) above. In these embodiments, administration begins sometime after the onset of ischemia, but preferably as close as possible to the onset of ischemia. For example, administration may begin approximately 5, 10, 15, 20, 25, or 30 minutes after the onset of ischemia. The timing of administration may or may not correspond to the onset of reperfusion.

[0074] In other embodiments, the method may include an additional step of applying NO containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), immediately followed by the administration of NO containing 20 ppm or less. x A gaseous composition. For example, a loading dose may be administered for about 10 minutes, about 15 minutes, about 20 minutes, about 30 minutes, or about 10 minutes to about 30 minutes. In another example, a loading dose may be administered for about 30 minutes, about 35 minutes, about 40 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, or 30 minutes to 60 minutes. The two compositions may be the same except for the nitric oxide concentration. Alternatively, the two compositions may be different. The concentration of nitric oxide in the loading dose may be decreased during the loading period until the nitric oxide concentration is 20 ppm or lower. The rate of decrease may be constant or not. Alternatively, the concentration of nitric oxide in the loading dose may be kept constant during the loading period and then decreased to a nitric oxide concentration of 20 ppm or lower. In a preferred embodiment, the two compositions are the same, and the composition is an infusion solution, preferably a cell-free infusion solution. In other embodiments, the cell-free perfusion solution is Steen Solution™, which optionally contains sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin. In an exemplary embodiment, the organ is the heart, lung, or kidney.

[0075] Example

[0076] The following examples illustrate various repetitions of the present invention.

[0077] Example 1: Lung Transplantation Protocol

[0078] Based on modified standard lung donor inclusion criteria, the study included a total of 20 lungs (8 lungs with gNO and perfusion fluid, 8 lungs with perfusion fluid only, and 4 lungs with ventilated gNO and perfusion fluid). Triple-hand studies included gNO plus perfusion fluid, perfusion fluid only, and ventilated gNO and perfusion fluid (preliminary study). The XVivo perfusion device system with cell-free perfusion fluid (Steen solution) will be used. The maximum cold ischemia time for the lungs will be 8–10 hours. The duration of ex vivo lung perfusion will be up to 12 hours. Lung health will be assessed through a grading system, biomarker evaluation, and histopathological evaluation.

[0079] Hierarchical system

[0080] The grading system comprises a 0-10 grade (total score) based on a comprehensive measurement using three variables. The three variables are: 1) δPaO2, weighted 0-4 using four categories: 0 = <350 mmHg; 1 = ≥350-<400 mmHg; 2 = ≥400-<450 mmHg; 3 = ≥450-<500 mmHg; 4 = ≥500 mmHg; 2) Static lung compliance, weighted 0-4 (change from baseline): 0 = no improvement or worsening of compliance; 1 = 1-3% improvement; 2 = 4-7% improvement; 3 = 8-11% improvement; and 4 = 12-15% improvement in compliance; and 3) Pulmonary vascular resistance (PVR), weighted 0-2: 0 = no change or increase in PVR; 1 = 1-7% decrease in PVR; 2 = 8-15% decrease in PVR.

[0081] Biomarker assessment

[0082] Damage assessment molecular proteins (DAMPs), high-mobility group box-1 (HMGB1), S100A8 (MRP8, cadherin A), S100A9 (MRP14, cadherin B), and serum amyloid A (SAA) will be the biomarkers evaluated. Cytokines of interest include TNF α-1, IL-1-β, IL-6, NLRP3, IL-10, and donor cell-free DNA.

[0083] Histopathological assessment

[0084] The histopathological parameters to be evaluated are evidence of interstitial and alveolar edema, hyaline membrane formation, and vascular integrity / damage (CD31 staining).

[0085] Donor lung retrieval

[0086] Current clinical practice after organ retrieval from a donor involves cryostatic preservation before transplantation to the recipient. During retrieval, the lungs are cold-flushed using a low-potassium dextran preservation solution, while simultaneously undergoing local cooling and ventilation. The lungs are then transported in a statically inflated state at 4°C. In the face of ischemia, hypothermia reduces metabolic activity and maintains cellular viability, thereby essentially slowing the cell death process (metabolic rate is 5% at 37°C). Therefore, cryopreservation is the main component of existing lung preservation techniques. However, the significant decline in organ metabolic function precludes the possibility of meaningful lung assessment and recovery.

[0087] The donor lung retrieval technique used in this study was as follows: Bronchoscopy and median sternotomy were performed. The pericardium and multiple cavities were opened. Both lungs were re-expanded and PO2 was assessed upon 100% FiO2 stimulation. Systemic heparinization was performed. A purse-string suture was made over the main pulmonary artery (PA). The PA was cannulated through the purse-string suture. The cannula was de-aired and connected to a de-aired Perfadex bag (the Perfadex bag should be deflated only by gravity (without pressurization), and the bag should not be more than 1 meter above the lung). 500 mcg of alprostadil was administered directly to the main PA. The superior vena cava (SVC) was ligated, the left and right atria were emptied, and the aorta was occluded.

[0088] Administer 4 liters of Perfadex antegradely. Ensure easy and rapid drainage of effluent from the right and left atria. Place localized ice on both lungs (however, use only one lung per cycle). Maintain ventilation with room air FiO2 at a tidal volume of 4–6 ml / kg / min and a respiratory rate of 10 breaths per minute. Once the infusion is complete, remove the heart. If the heart is being transplanted, incise the PA at the bifurcation. Do not separate the branch PAs. If the heart is not being transplanted, incise the PA at the RVOT immediately adjacent to the pulmonary valve. Ensure the left atrial cuff remains intact. Administer 2 liters of cold Perfadex retrogradely via the pulmonary vein. Bilaterally incise the pericardium at the diaphragmatic level and completely separate the pericardium from the diaphragm. Bilaterally incise the inferior pulmonary ligaments. Dissect the posterior pericardium cephalicly from the posterior mediastinum to the carina. Dissect the trachea cephalicly to the level of the cricoid cartilage. Retract the endotracheal tube back to the level of the larynx. Inflate the lungs to 50% of total lung capacity at 50% FiO2. Separate the trachea using two nails under load above the cricoid cartilage. Dissect the posterior trachea from the caudal side of the posterior mediastinum (esophagus) towards the diaphragm. Dissect the left PA from the aortic arch and separate the aortic valve. Remove the lungs and store them in ice-cold Perfadex solution. If a heart has been harvested for transplantation, remove 10 cm of the donor descending aorta and place it in the same cold Perfadex solution bag as the lungs. This will be used later for pulmonary artery reconstruction.

[0089] Ex vivo lung perfusion (EVLP)

[0090] The main principle of EVLP involves perfusing and ventilating a lung outside the body within a closed container that maintains the temperature, humidity, and sterility of the donor organ. The EVLP circuit includes a centrifugal pump that circulates the perfusion fluid, passing it through a membrane gas exchanger and a leukocyte filter before it enters the pulmonary artery (PA). This system is very similar to those used in extracorporeal circulation circuits (ECC) for cardiac surgery. Cell-free Steen solution will be used as the perfusion fluid for the EVLP procedure.

[0091] In simple terms, the EVLP process is as follows: The lungs are placed in a special covered plastic chamber to hold them in a stable position and provide a warm, humid environment during ventilation. Infusing fluid (propelled by a centrifugal pump) is delivered to the lungs through a cannula located in the pulmonary artery (PA). Return from the lungs is passively transported through the pulmonary veins (PV) by gravity, and the infusing solution is collected in a reservoir and then recirculated via a pump and a membrane oxygenator. A gas oxygenator is connected to a tank containing a special gas mixture of oxygen (6%), carbon dioxide (8%), and nitrogen (86%). The ventilator delivers an airway to the lungs and is connected to a tracheothoracic tube.

[0092] The lung graft (cooled during organ retrieval and subjected to static cold preservation) is gradually rewarmed over a 45-minute period. Lung perfusion begins at a low flow rate (.10–.15 L / min) and is then gradually increased during rewarming until the volume delivered in the pulmonary artery is 40–50% of the estimated cardiac output (70 L / kg / min). Because ischemia-reperfusion injury (IRI) after organ retrieval weakens the capillary-alveolar barrier, and because IRI impairs its integrity, increases the permeability of the alveolar-capillary membrane, and can lead to pulmonary edema, PA pressure needs to be maintained at a low level (<15–20 mmHg).

[0093] During the rewarming phase, the membrane oxygenator delivers an O2 supply to the graft, followed by pCO2 and pH values ​​close to those typically measured in the PA. The catheter remains in situ, continuously measuring PA and left atrial (LA) pressure throughout the EVLP process. Mechanical ventilation of the lungs is initiated when the perfusion fluid temperature reaches 32 degrees Celsius (typically approximately 30 minutes after perfusion begins).

[0094] Below are the 19 steps of the EVLP process. This is a quick reference guide, not an exhaustive plan.

[0095] Step 1. Transport the lungs to XPS. Comply with UNOS standards.

[0096] Step 2. Intubate the lung. Trim the LA cannula (green) to approximate the atrial opening and suture it with continuous polypropylene sutures. For the PA cannula (yellow), open the PA lumen and insert the XVIVO cannula. If pulmonary artery reconstruction is required, suture a segment of the donor descending aorta to the pulmonary artery and then intubate as described above. Secure the cannula in place through the plexus using an umbilical cord or ribbon, and re-tie it to the cannula below the pressure line. Intubate, clamping the trachea to prevent deflation. Insert the ET tube into the trachea and secure it in place with an umbilical cord or ribbon.

[0097] Step 3. Backflush. Backflush one liter of cold Perfadex, checking for leaks around the cannula. Keep the ET tubing clamped.

[0098] Step 4. System Setup. Establish the infusion circuit on the XPS using PGM. Open the dome by removing the plastic, then the first layer of blue packaging material on the back table. Place a clear U-shaped cover on the XPS top bar above the blue table and secure it to the XPS with clips, covering the blue table so that the "U" opening of the U-shaped cover is on the right side, allowing the tubing to pass through. Aseptically open the second layer of blue dome on the blue table of the Xvivo machine. Connect the red 3 / 8-inch drain line to the back of the dome and wrap 1 / 4 of the line around the outside of the post and into the red roller pump. Switch the drain bag and connect the orange and red lines to the bag. Add the return line from the top of the reservoir to the stopcock valve. Close the stopcock valve on the red line to the reservoir and open it to the drain bag. Set the sensor and flush with sterile saline, add the blue line for the heater / cooler, and then connect the intravenous gas line to the back of the Quadrox. Perform a check to ensure sufficient O2 and tri-gas levels.

[0099] Step 5. System Cleaning. Add 1500 ml of STEEN to the reservoir, add the medications (heparin 10,000 units, methylprednisolone 500 mg, ceftazidime 1 gm), first turn on the Cardiohelp and heater cooler (set to 23), then turn on the UPS, followed by the touchscreen and ventilator. Clean the perfusion circuit by removing the yellow cap on the back of the Quadrox, any transparent caps on the reservoir, and the blue cap on the white blood cell filter. Increase the Cardiohelp to 1000 RPM and check the circuit for leaks. After one minute, increase the Cardiohelp to 3000 RPM. After a few minutes, increase the Cardiohelp to 5000 RPM and run it for one minute, checking to ensure all air has exited the tubing, and reduce the RPM to below 250.

[0100] Step 6. Data Setup Log. On the touchscreen setup page, fill in the required information and change the pH value to an acceptable alarm level (6.8). On the service page, click the green PGM Calibration button and fill in the pH and PO2 values ​​based on the calibration numbers found on the PGM package. Set the timer on the main screen (1 and 2 are incrementing counts, and #3 is decrementing counts (set #3 to 10 minutes)). On the ventilator, press Setup, Mode (S) CMV, and press Confirm. Enter the parameters from the spreadsheet and press Confirm. Also set the alarm parameters on the flowchart. On the I / O panel on the back of the XPS, place the inspiratory port on the right side of the lung circuit and the expiratory port (blue tip) on the left side of the lung circuit. Connect the flow sensor tubing, the transparent one to the white one, and the blue one to the blue one. Connect the intravenous gas mixing tubing. Connect the high-pressure O2 tubing. Run the humidifier filter on the lung end of the circuit between the flow sensor and the ET-T. Perform the preoperative check, press System, then Test and Calibrate. Select each calibration test one at a time. Follow the tightness and steps, follow the flow sensor and steps, O2 cells do not need to be tested.

[0101] Step 7. Retrograde Flushing. Redirect the flow in the perfusion circuit using a sterile clamp, allowing flow to flow into the LA and out of the PA. Start at 750 RPM and slowly increase the RPM until there is sufficient flow to fill the PA cannula and flush the lung until there is no blood in the STEEN solution, which should be approximately 250 cc. Use a circulation pump to direct this STEEN solution into a waste bag. Once there is no blood, connect the cannula and circuit on the LA side to ensure the lung is filled with STEEN solution. There should be enough STEEN to pass through the LA cannula and fill the PA cannula, then reset the direction of the circulation pump to direct it into the reservoir. Reposition the sterile clamp on top of the PA cannula and connect it to the circuit. Clamp bridge (this should be the only clamp).

[0102] Step 8. Calibration. Calibrate the perfusion flow sensor and pressure sensor. (The flow sensor calibration can be performed before retrograde with the lung placed in the circuit). The pressure sensor calibration should be performed immediately after retrograde, with the lung in the circuit (the fluid level top (where the cannula connects to the lung) and the pressure stopcock valve should be at the same level, and the syringe should be removed from the pressure sensor tubing to fill the STEEN, ensuring the stopcock valve moves to cut off the saline solution).

[0103] Step 9. Proceeding. Start Timer 1 (Injection Timer) and set the first hour according to the EVLP worksheet.

[0104] Step 10. First Hour Intensive. The Cardiohelp and heater cooler will be adjusted every 10 minutes until each reaches its maximum value. Ventilation cannot begin until the temperature reaches 32°C. Ventilation can begin at 32°C. Three-gas purging must be initiated simultaneously with ventilator startup.

[0105] Step 11. Lung recruitment. Do not confuse this with O2 stimulation. Lung recruitment involves manually holding the expiratory button on the ventilator for 15 seconds. This button should be pressed after inhalation to maintain exhalation.

[0106] Step 12. O2 stimulation. O2 stimulation will only involve changing the settings on the ventilator, following the recruitment settings on the EVLP worksheet. (Multiply by IBW x 10 to get Vt, increasing FiO2 to 100%, BPM to a maximum of 10)

[0107] Step 13. Aspiration. During the last minute of O2 excitation, arterial and venous perfusion samples should be aspirated from the sensors. Click the gray pH button on the main screen to lock the gas value. (When the results return from the lab, perform precise pH calibration on the LA and PA.) Record the appropriate information, then reset to normal settings on the ventilator (at this point, set the alarm and ventilator to high).

[0108] Step 14. X-ray. After O2 excitation, an X-ray should be taken for benchmark comparison with future X-rays.

[0109] Step 15. Steen Dilution. STEEN will need to be diluted after re-expansion, excitation, aspiration, and X-ray (or during X-ray). Open the + / - pump control window on the touchscreen. Touch the "Remove" button and slide your finger down until the removal button is locked (if using the clamp method, replace the clamp). Observe the fluid level in the reservoir drop. Once enough STEEN has been removed, touch the "Remove" button to close it (if using the clamp method, replace the clamp). Add 3 or 4 vials of fresh STEEN. Another dose of medication may also be added.

[0110] Step 16. Perform lung maintenance and assessment over the next 3–5 hours. Repeat steps 12 and 13 at the 50-minute mark every hour (minus precise calibration).

[0111] Step 17. About one hour before the lung is removed for transplantation, repeat step 14. Comparing the first and second X-rays will help determine if the lung is suitable for transplantation.

[0112] Step 18. Repeat step 15 after 6 hours of perfusion.

[0113] Step 19. Rapid Cooling. After consent is given for lung transplantation, set the heater / cooler to 15°C. At 32°C, when the lung is 50% inflated, clamp the ET tube, disconnect the lung from the perfusion circuit, and flush with two liters of cold Perfadex. Place the lung in a sterile bag containing Perfadex. (Similar to the standard donor procuring protocol).

[0114] Step 20. Clean and store the XPS. Wipe the XPS surface with disinfectant, store it with the latches open, and plug it into a socket to charge. Evacuate the system every 10 minutes after activation.

[0115] For reference, please see Figure 1 and Figure 2 It shows a possible system schematic diagram.

[0116] Example 2. In a blinded, randomized, controlled pediatric asphyxia model of cardiac arrest, inhaled nitric oxide improved brain mitochondrial function.

[0117] introduction

[0118] Neurological injury following cardiac arrest (CA) remains common in children. Inhaled nitric oxide (iNO) can alleviate mitochondrial dysfunction in the brain induced by CA (a key focal point of secondary brain injury). It is hypothesized that, compared with placebo, animals treated with 20 ppm of iNO during CPR and within four hours after spontaneous circulation recovery (ROSC) following asphyxia and cardiac arrest will have improved cerebral blood flow (CBF) and improved mitochondrial function, as defined by increased respiratory control ratio and reduced mitochondrial reactive oxygen species (mtROS).

[0119] method

[0120] Four-week-old pigs underwent 7 minutes of asphyxia followed by ventricular fibrillation. Guided CPR was performed with a compression depth (CD) ≥ 1 / 3 of the thoracic diameter, and standard epinephrine was administered for 10 minutes or until ROSC, followed by standardized post-ROSC care. Subjects were randomly assigned in a blinded manner (1 minute after the start of CPR, starting with iNO 20 ppm, n=10, or placebo, n=10). A sham group (n=4) did not experience CA or CPR. Benchmark and continuous CBF measurements were performed using invasive clinical and non-invasive optical instruments. Cortical and hippocampal tissues were analyzed using high-resolution respiratory measurements to assess mitochondrial function. T-tests and ANOVA were used where applicable. Longitudinal hemodynamic variables were compared using generalized estimating equations to control for intra-subject correlations.

[0121] result

[0122] Seven out of ten animals in the placebo group and ten out of ten animals in the iNO group survived (p=0.21). There were no significant differences in invasive or non-invasive CBF between the treatment groups during CPR and after ROSC. In animals treated with iNO, cortical and hippocampal RCR were significantly increased (p=.04, 0.007), and mtROS production was significantly decreased (p<0.001, p=0.03). There were no differences in systemic or pulmonary hemodynamics between the iNO and placebo groups, but iNO-treated animals showed a trend towards lower mean pulmonary artery pressure during CPR (28.1±9.8 vs. 42.6±6.0, p=0.14). iNO preserved brain mitochondrial function (increased RCR) and limited mtROS production in a porcine model of pediatric CA. Further research is needed to evaluate this potential neuroprotective effect of iNO against ischemia-reperfusion injury and cardiac arrest.

[0123] This disclosure relates to the following implementation plan:

[0124] 1. A method for improving the viability of an organ intended for transplantation, the method comprising the continuous direct administration of NO containing 20 ppm or less to the organ via an organ perfusion system or ventilation. x A composition of gases.

[0125] 2. The method of claim 1, wherein the composition is applied for at least one hour but not more than 12 hours.

[0126] 3. The method of claim 1 or 2, wherein the composition is administered after the organ has been obtained from the donor.

[0127] 4. The method of claim 1 or 2, wherein the composition is administered while the organ is in a brain-dead donor body.

[0128] 5. The method of any one of claims 1 to 3, wherein the organ is selected from the group consisting of the lung, heart, liver, kidney, pancreas, intestine, thymus and cornea.

[0129] 6. The method of any one of claims 1 to 5, wherein the NO x The amount is approximately 10 ppm to approximately 15 ppm.

[0130] 7. The method of any one of items 1 to 5, wherein the NO x The amount is approximately 5 ppm to approximately 10 ppm.

[0131] 8. The method of any one of items 1 to 5, wherein the NO x The amount is from about 1 ppm to about 5 ppm.

[0132] 9. The method of any one of claims 1 to 8, wherein the composition is an organ perfusion solution and optionally further comprises one or more additional compounds.

[0133] 10. The method of claim 9, wherein the perfusion solution is a cell-free perfusion solution.

[0134] 11. The method of any one of claims 1 to 10, wherein the method further comprises administering NO to the organ containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), immediately followed by the administration of NO containing 20 ppm or less. x The gaseous composition.

[0135] 12. The method of claim 11, wherein the loading dose composition is an infusion solution.

[0136] 13. The method of claim 12, wherein the perfusion solution is a cell-free perfusion solution.

[0137] 14. The method as described in any one of items 1 to 13, wherein NO x The total application time of the gas shall not exceed 12 hours.

[0138] 15. The method of any one of items 1 to 14, wherein mitochondrial function is significantly preserved.

[0139] 16. The method of any one of items 1 to 15, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared with an untreated control.

[0140] 17. A method for improving the viability of organs damaged by ischemia-reperfusion, the method comprising continuously administering, directly to the organ via an organ perfusion system, a solution containing 20 ppm or less of NO. x A composition of gases.

[0141] 18. The method of claim 17, wherein the composition is applied for at least one hour but not more than 12 hours.

[0142] 19. The method of claim 17 or 18, wherein the organ is used for transplantation and the composition is administered after the organ is obtained from a donor.

[0143] 20. The method of claim 17 or 18, wherein the organ is used for transplantation and the composition is administered while the organ is in a brain-dead donor body.

[0144] 21. The method of any one of items 17 to 20, wherein the organ is selected from the group consisting of the lung, heart, liver, kidney, pancreas, intestine, thymus and cornea.

[0145] 22. The method of any one of items 17 to 21, wherein the NO x The amount is approximately 10 ppm to approximately 15 ppm.

[0146] 23. The method of any one of items 17 to 21, wherein the NO x The amount is approximately 5 ppm to approximately 10 ppm.

[0147] 24. The method of any one of items 17 to 21, wherein the NO x The amount is from about 1 ppm to about 5 ppm.

[0148] 25. The method of any one of claims 17 to 23, wherein the composition is an organ perfusion solution and optionally further comprises one or more additional compounds.

[0149] 26. The method of claim 25, wherein the perfusion solution is a cell-free perfusion solution.

[0150] 27. The method of any one of claims 17 to 26, wherein the method further comprises administering NO to the organ containing about 20 ppm to about 40 ppm. x The gaseous composition (“loading dose”) is administered for up to about 1 hour (“loading period”), immediately followed by the administration of NO containing 20 ppm or less. x The gaseous composition.

[0151] 28. The method of claim 27, wherein the loading dose composition is an infusion solution.

[0152] 29. The method of claim 28, wherein the perfusion solution is a cell-free perfusion solution.

[0153] 30. The method of any one of items 17 to 29, wherein NO x The total application time of the gas shall not exceed 12 hours.

[0154] 31. The method of any one of items 17 to 30, wherein mitochondrial function is significantly preserved.

[0155] 32. The method of any one of items 17 to 31, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared with an untreated control.

[0156] 33. A transplantation method, the method comprising:

[0157] (a) Continuous administration of NO containing 20 ppm or less directly to the organ to be transplanted via an organ perfusion system. x Compositions of gases; and

[0158] (b) Transplant the organ into the recipient.

[0159] 34. The method of claim 33, wherein the composition is applied for at least one hour but not more than 12 hours.

[0160] 35. The method of claim 33 or 34, wherein the composition is applied after the organ has been obtained from the donor.

[0161] 36. The method of claim 33 or 34, wherein the composition is administered while the organ is in a brain-dead donor body.

[0162] 37. The method of any one of items 33 to 36, wherein the organ is selected from the group consisting of the lung, heart, liver, kidney, pancreas, intestine, thymus and cornea.

[0163] 38. The method of any one of items 33 to 37, wherein the NO x The amount is approximately 10 ppm to approximately 15 ppm.

[0164] 39. The method of any one of items 33 to 37, wherein the NO x The amount is approximately 5 ppm to approximately 10 ppm.

[0165] 40. The method of any one of items 33 to 37, wherein the NO x The amount is from about 1 ppm to about 5 ppm.

[0166] 41. The method of any one of claims 33 to 40, wherein the composition is an organ perfusion solution and optionally further comprises one or more additional compounds.

[0167] 42. The method of claim 41, wherein the perfusion solution is a cell-free perfusion solution.

[0168] 43. The method of any one of claims 33 to 42, wherein the method further comprises administering NO to the organ containing about 20 ppm to about 40 ppm. x The gas composition (“load dose”) is applied for up to about 1 hour (“load period”), after which step (a) is performed immediately.

[0169] 44. The method of claim 43, wherein the loading dose composition is an infusion solution.

[0170] 45. The method of claim 44, wherein the perfusion solution is a cell-free perfusion solution.

[0171] 46. ​​The method of any one of items 33 to 45, wherein NO x The total application time of the gas shall not exceed 12 hours.

[0172] 47. The method of any one of items 33 to 46, wherein mitochondrial function is significantly preserved.

[0173] 48. The method of any one of items 33 to 47, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared with an untreated control.

Claims

1. A method for improving the viability of an organ intended for transplantation, the method comprising: The solution will contain cell-free perfusion solution and 40 ppm NO. x A loading dose of gas is applied to the organ; as well as Continuous administration of a cell-free perfusion solution and approximately 0.05 ppm to approximately 20 ppm NO to the organ via an organ perfusion system. x Gas composition, The composition is administered immediately after the loading dose is applied.

2. The method of claim 1, wherein the composition is applied for at least one hour but no more than 12 hours.

3. The method of claim 1 or claim 2, wherein the composition is administered after the organ has been obtained from the donor.

4. The method of claim 1 or claim 2, wherein the composition is administered while the organ is in a brain-dead donor body.

5. The method according to any one of claims 1 to 3, wherein the organ is selected from the group consisting of the lung, heart, liver, kidney, pancreas, intestine, thymus and cornea.

6. The method according to any one of claims 1 to 5, wherein the NO x The amount is approximately 10 ppm to approximately 15 ppm.

7. The method according to any one of claims 1 to 5, wherein the NO x The amount is approximately 5 ppm to approximately 10 ppm.

8. The method according to any one of claims 1 to 5, wherein the NO x The amount is from about 1 ppm to about 5 ppm.

9. The method of any one of claims 1 to 8, wherein the cell-free perfusion solution comprises sodium caprylate, N-acetyl-DL-tryptophan and human serum albumin.

10. The method of any one of claims 1 to 9, wherein the NO in the loading dose x The concentration was decreased during the loading period until continuous administration of a cell-free perfusion solution and 20 ppm or less of NO was continued. x The gaseous composition.

11. The method of any one of claims 1-9, wherein the NO in the loading dose x The concentration was kept constant during the loading period, and then when a cell-free perfusion solution containing 20 ppm or less NO was administered... x The gas composition is reduced.

12. The method according to any one of claims 1 to 11, wherein NO x The total application time of the gas shall not exceed 12 hours.

13. The method of any one of claims 1 to 12, wherein mitochondrial function is significantly preserved.

14. The method of any one of claims 1 to 13, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared with an untreated control.

15. A method for improving the viability of organs damaged by ischemia-reperfusion, the method comprising: The solution will contain cell-free perfusion solution and 40 ppm NO. x A loading dose of gas is applied to the organ; as well as Direct and continuous administration of a cell-free perfusion solution and approximately 0.05 ppm to approximately 20 ppm of NO directly into the organ. x A gaseous composition, wherein the cell-free perfusion solution and NO are administered using an organ perfusion system. x The gas was applied directly to the organ; and The composition is administered immediately after the loading dose is applied.

16. The method of claim 15, wherein the composition is applied for at least one hour but no more than 12 hours.

17. The method of claim 15 or claim 16, wherein the organ is used for transplantation, and the composition is administered after the organ is obtained from a donor.

18. The method of claim 15 or claim 16, wherein the organ is used for transplantation, and the composition is administered while the organ is in a brain-dead donor body.

19. The method of any one of claims 15 to 18, wherein the organ is selected from the group consisting of the lung, heart, liver, kidney, pancreas, intestine, thymus, and cornea.

20. The method of any one of claims 15 to 19, wherein the NO x The amount is approximately 10 ppm to approximately 15 ppm.

21. The method of any one of claims 15 to 19, wherein the NO x The amount is approximately 5 ppm to approximately 10 ppm.

22. The method of any one of claims 15 to 19, wherein the NO x The amount is from about 1 ppm to about 5 ppm.

23. The method of any one of claims 15 to 21, wherein the cell-free perfusion solution comprises sodium caprylate, N-acetyl-DL-tryptophan, and human serum albumin.

24. The method of any one of claims 15 to 23, wherein the NO in the loading dose x The concentration was decreased during the loading period until continuous administration of a cell-free perfusion solution and 20 ppm or less of NO was continued. x The gaseous composition.

25. The method of any one of claims 15-23, wherein the NO in the loading dose x The concentration was kept constant during the loading period, and then when a cell-free perfusion solution containing 20 ppm or less NO was administered... x The gas composition is reduced.

26. The method of any one of claims 15 to 25, wherein NO x The total application time of the gas shall not exceed 12 hours.

27. The method of any one of claims 15 to 26, wherein mitochondrial function is significantly preserved.

28. The method of any one of claims 15 to 27, wherein mitochondrial reactive oxygen species (mtROS) are reduced in the organ compared with an untreated control.

Images